Chimeric vector and preparation method and use thereof

ABSTRACT

A chimeric vector is provided in the present invention, which is formed by ligating a Vif protein and a functional protein, the functional protein being a Raf protein or a Rev protein. By designing and constructing a Rev-Vif-C vector and then demonstrating that the Rev-Vif-C vector has a good anti-virus effect by a variety of experiments, the present invention proposes a novel anti-virus technology against the Rev protein of HIV-1. Moreover, by designing and constructing a RBD-Vif-C vector and then demonstrating that the RBD-Vif-C vector has a good tumor cell killing effect by cell-level experiments in vitro and experiments in vivo with nude mouse tumor models, the present invention proposes a novel anti-tumor technology specifically against mutant KRAS.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a 371 application of the International PCTapplication serial no. PCT/CN2014/080199, filed on Jun. 18, 2014. Theentirety of the above-mentioned patent application is herebyincorporated by reference herein and made a part of this specification.

TECHNICAL FIELD

The present invention relates to a protein anti-cancer medicine, andmore specifically, relates to a chimeric vector, and preparation methodand use thereof.

BACKGROUND

All this time, if change happens in cell genome which controls theexpression or the function in cell growth and cell differentiation, itis considered as the major cause inducing tumor. Molecular biology studyin tumor is directed to confirm those changes of genes in various tumortypes and to illustrate the functions of these genes in tumorigenesis.In particular, RAS gene is one of the most common gene families in tumormutation of human.

Under normal physiology circumstances, when a signaling pathway such asEGFR is activated after cells are stimulated by the external, thewild-type KRAS is phosphorylated by a tyrosine kinase such as activeEGFR, before being transiently activated. The activated KRAS canactivate the downstream signaling protein in the signaling pathway,after that the KRAS is inactivated rapidly. The activation/inactivationeffect of the KRAS is controllable. However, mutant KRAS protein causesa dysfunction of protein that the mutant KRAS is still under activatedstatus without the stimulation of activation signal from EGFR, such thatthe functional status of mutant KRAS is uncontrollable which makes thetumors proliferate continuously. The RAS gene, like a “switch” in vivo,plays an important regulating role in the signal transduction path ofprocesses such as tumor cell growth and angiogenesis. The encodedprotein of normal KRAS gene can inhibit tumor cell growth. Once the KRASgene mutates, it will continuously stimulate the cell growth,disorganize the growth rhythm and thus cause tumorigenesis. Because themutation of the KRAS gene generally occurs at the early stage of thetumor malignancy, also the KRAS genes of the primary tumor and themetastases are highly consistent; and it is generally acknowledged thatthe status of KRAS gene will not change with the treatment. Therefore,the detection of the mutation in the KRAS gene is an important indicatorfor in-depth understanding of the condition of oncogene as well as thedevelopment, prognosis and the curative effect of the chemoradiotherapyfor various cancers, having a vitally important clinical significance.

In China, pancreatic cancer has always been one of the top ten malignanttumors that causing the population death, having a five-year survivalrate of less than 5%, being one of the worst malignant tumors inprognosis. In recent years, colorectal cancer has become the secondbiggest cancer killer in Guangdong area with obviously increasing trend,having a morbidity and a mortality that significantly increase year byyear. The RAS gene of tumor cells has a mutation rate of about 25% whilepancreatic cancer, colorectal cancer and non-small alveolar lung cancerhave a mutation rate of 90%, 45% and 35%, respectively.

In late 1970s and early 1980s, a disease with a dysfunction of theimmune system as a major characteristic arose in Europe and the UnitedStates. Afterwards, scientists from various countries started to explorethe nosogenesis and the therapeutic schedule for such disease. Until1983, after the Pasteur Research Group in France first successfullyisolated this new retrovirus, the theoretical study and the medicaltreatment for HIV-1 became more and more. Nowadays, medicine againstHIV-1 mainly acts on different stages of the life cycle of the virus,and specifically on some necessary enzymes such as reverse transcriptaseand protease.

Although there are many anti-HIV-1 medicines that are commercialavailable nowadays and HARRT is widely used, the resulting problems suchas drug resistance, huge medical expenses and the side effects of themedicines should not be underestimated. Plasma viral load in aconsiderable number of patients can be reduced below a detectable levelby highly active antiretroviral therapy (HARRT), but the rebound afterstopping taking such medicine and the severe toxic side effects arestill can't be solved. Gene therapy has shown its potential foranti-virus and some research achievement has entered clinical teststage, but its low efficiency and the adverse reaction possibly broughtby the foreign vector are still the major obstacle to research anddevelopment. Nowadays, there are four major hot spots for the researchand development of anti-HIV-1 medicine internationally: 1) inhibitorthat inhibits virus entering into the cells; 2) neutralizing antibody;3) integrase inhibitor; and 4) chemical chemokine receptor antagonists.Scientists still keep trying to explore an anti-virus medicine that issafer, more effective and more affordable.

Regulator of expression of virion proteins (Rev) is an indispensableregulatory protein in the transcription process of HIV-1. The Revinteracts with RRE of mRNA of the virus so as to aid unspliced orpartially spliced mRNA of HIV-1 to transfer out of the nucleus. If theexpression of the Rev is inhibited, the unspliced or partially splicedmRNA of HIV-1 will be unable to transfer out of the nucleus, leading toa complete degradation within the nucleus, and a further block of thereplication of HIV-1. Therefore, how to inhibit the expression of Revprotein will be an important target for research and development ofanti-HIV-1 medicine.

SUMMARY OF THE INVENTION

One of the objectives in the present invention is to invent a medicinethat uses Vif in HIV-1.

First a chimeric vector is provided, which comprises nucleic acidencoding a Vif protein and a functional protein. The functional proteinis a Raf protein or a Rev protein.

An end of the functional protein is ligated to the Vif protein.

The chimeric vector is obtained by replacing nucleic acid encoding anN-terminus of the Vif protein with nucleic acid encoding a bindingdomain of an N-terminus of the Raf protein which can specifically bindto GTP-Kras. Such chimeric vector is named as Rev-Vif-C.

The chimeric vector is obtained by replacing nucleic acid encoding theN-terminus of the Vif with nucleic acid encoding a multimerizationdomain of the Rev. Such chimeric vector is named as RBD-Vif-C.

Further a method for the production of a fusion protein comprising Vifand Revof, the method comprising steps as follows:

a) preparing a chimeric vector, comprising respectively replacingnucleic acid encoding an amino acid sequence at positions 1-79 (SEQ IDNO: 5) of an N-terminus of a Vif protein with nucleic acid encoding twooligomerization domains which are provided on a protein structure of aRev, thus three new chimeric vectors ROL1-Vif-C, ROL2-Vif-C andROL12-Vif-C are constructed, the ROL1-Vif-C comprising nucleic acidencoding an oligomerization domain of a Rev N-terminus, the ROL2-Vif-Ccomprising nucleic acid encoding an oligomerization domain of a RevC-terminus, the ROL12-Vif-C comprising nucleic acid encoding twooligomerization domains of the Rev N-terminus and the Rev C-terminus,

the oligomerization domain of the Rev N-terminus is amino acids atpositions 9-26 (SEQ ID NO: 6) of an amino acid sequence of the Revprotein,

the oligomerization domain of the Rev C-terminus is amino acids atpositions 51-65 (SEQ ID NO: 7) of the amino acid sequence of the Revprotein,

the two oligomerization domains of the Rev N-terminus and the RevC-teiminus are amino acids at positions 9-65 (SEQ ID NO: 8) of the aminoacid sequence of the Rev protein,

and a Vif-C moiety is amino acids at positions 80-193 (SEQ ID NO: 9) ofthe amino acid sequence of the Vif protein;

b) cloning these three chimeric vectors into an expression vectorpcDNA3.1 respectively wherein, sequences of the three vectors are shownas SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and enzyme cutting sitesused for ligating the vectors to the pcDNA3.1 are bamH I and Xhol I;

c) performing a transient expression by transfecting a host cell withthe three vectors mentioned above;

d) culturing the host cell under conditions that the fusion protein isproduced; and

e) isolating the fusion protein from the host cell culture.

Also a method for the production of a fusion protein comprising Vif andRaf, the method comprising steps as follows:

a) preparing a chimeric vector, comprising respectively replacingnucleic acid encoding replacing an amino acid sequence at positions 1-79of an N-terminus of a Vif protein with nucleic acid encoding a RASbinding domain RBD which is provided on a protein structure of a Raf andspecifically binds to GTP-KRAS, thus a new chimeric vector of RBD-Vif-Cis constructed;

b) cloning the chimeric vector into an expression vector pcDNA3.1 andperforming a transient expression by transfecting a host cell with thevector mentioned above;

c) ligating nucleic acid encoding a transmembrane peptide segment PTD toan Escherichia coli expression vector of pet-32a;

d) performing PCR amplification using the RBD-Vif-C as a template,ligating the RBD-Vif-C of the obtained PCR product to the expressionvector pet-32a, and the PTD being located on an N-terminus of theRBD-Vif-C to form a fusion expression vector PTD-RBD-Vif-C;

e) transforming plasmids of the PTD-RBD-Vif-C into the Escherichia coliBL21(DE3);

f) culturing the transformed Escherichia con; and

g) using a nickel column to pufify a single PTD-RBD-Vif-C protein fromthe Escherichia coli culture.

In particular, culturing mentioned in f) is to inoculate a single colonyof the transformed Escherichia coli to a LB liquid medium containingampicillin for culturing overnight; and then the colony is inoculated toa 37° C. preheated LB liquid medium containing ampicillin with a volumeratio of 1:50 for culturing until OD600 reaches 0.6; IPTG is added tothe expressed PTD-RBD-Vif-C protein until having a final concentrationof 0.4 mmol/L, and the bacteria is collected after induction.

A method of purification mentioned in g) is to wash total bacteria withPBS Buffer. After being resuspended and ultrasonic treated, a lysatesupernatant is obtained by a high speed centrifugation, and an elutedprotein is collected after an affinity chromatography purification usingthe nickel column.

Sequence of the fusion expression vector PTD-RBD-Vif-C is shown as SEQID NO:4.

Structures from the N-terminus to the C-terminus are transmembranepeptide PTD moiety, KRAS binding moiety of the RBD and degradationmoiety of the Vif-C, respectively.

Vif protein is an important protein of HIV-1 itself, can bind to targetproteins and ligate them to an E3 ligase complex (Vif-SCF-CUL5 complex).Then such complex will make the target proteins ubiquitinated, thusleading to the degradation of the proteins in a proteasome. The presentinvention mainly focuses on the corresponding cancers caused by themutation of KRAS, attempts to replace the N-terminus of the Vif with theRBD of the N-terminus of the Raf protein which can specifically bind tothe GTP-Kras, and by means of several tests in vitro and in vivo,explores the efficiency and the acting mechanisms for protein obtainfrom such chimeric type vector degrading the mutant KRAS. This technicalinvention has a very important significance for further researching anddeveloping an anti-tumor protein medicine. Thus, this new method ofconstructing the chimeric vector that is provided in the presentinvention will very likely be a new technology of anti-tumor withextremely significant academic value and practical value.

(1) In order to overcome the defect and deficiency in the prior art, theobjective of the present invention is to provide a method ofconstructing a new type of chimeric vector and use thereof in the lifescience research and the clinical treatment for tumor.

(2) A new tumor-killing technology corresponding to KRAS gene isprovided in the present invention.

(3) A new technology of degrading the expression of KRAS protein isprovided in the present invention.

(4) A new technology that can specifically degrade the protein inparticular condition or in particular modification on the protein levelis provided in the present invention.

(5) A new method of degrading multiple proteins is provided in thepresent invention, i.e. inserting a binding site of a certain protein tothe N-terminus of the Vif so as to realize the degradation process ofspecific protein by the ubiquitination path of the C-terminus of theVif.

(6) Use of a new technology for treating pancreatic cancer tumor isprovided in the present invention.

(7) Use of a new technology for treating large intestine cancer(colorectal cancer) tumor is provided in the present invention.

(8) Use of a new technology for treating lung cancer tumor is providedin the present invention.

As for another technical solution:

Vif protein is an important protein of HIV-1 itself, can bind to thetarget proteins and ligate them to an E3 ligase complex (Vif-SCF-CUL5complex). Then such complex will make the target proteins ubiquitinated,thus leading to the degradation of the proteins in proteasome. Mainlyaccording to the ubiquitination function of the Vif protein, the presentinvention replaces the N-terminus of the Vif with the multimerizationdomain of the Rev such that it can specifically bind to the Rev proteinof HIV-1 and then degrade the Rev by the ubiquitination function of theVif. Attack its shield with its own spears. Thus the objective ofinhibiting HIV-1 replication can be achieved with strong novelty andspecificity. Also it provides a new idea and method of researching anddeveloping the anti-HIV-1 medicine.

(1) In order to overcome the defect and deficiency in the prior art, theobjective of the present invention is to provide a method ofconstructing a new type of chimeric vector and use thereof in the lifescience research and the clinical treatment.

(2) A new method of protecting cells from the attack of HIV-1 isprovided in the present invention. After the CD4+T cells in the patientare sorted out for amplification in vitro, they are allowed to expressthe Rev-Vif-C vector stably and then transfused back into the patient.This can not only protect the patient from the second attack of HIV-1,but also help the patient clear the HIV-1 virus in the body.

(3) A new vector against the spontaneous mutation of HIV-1 is providedin the present invention. The drug resistance of HIV-1 is mostly due toits spontaneous mutation caused by the drugs. But the provided chimericvector is constructed by using the mutual combination of the domains ofthe Rev itself. It can avoid various possible mutations, and thus canpresent an excellent inhibitory effect on multiple mutant strains ofHIV-1.

(4) A new technology of degrading the expression of the Rev protein isprovided in the present invention.

(5) A new technology of degrading multiple proteins is provided in thepresent invention, i.e. inserting a binding site of a certain protein tothe N-terminus of the Vif so as to realize the degradation process ofspecific protein by the ubiquitination pathway of the C-terminus of theVif.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the construction model of the chimeric vectorRBD-Vif-C.

FIG. 2 illustrates that the protein encoded by the chimeric vectorRBD-Vif-C can degrade the KRAS protein and inhibit its signaling pathwayof phosphorylation in the downstream.

FIG. 3 illustrates that the protein PTD-RBD-Vif-C has a good killingeffect on the tumor cells in vitro.

FIG. 4 illustrates that the protein PTD-RBD-Vif-C has a good anti-tumoreffect in mice.

FIG. 5 illustrates that the acute toxicity test of the proteinPTD-RBD-Vif-C in mice.

FIG. 6 illustrates the construction model of the chimeric vectorRev-Vif-C.

FIG. 7 illustrates that the protein encoded by the chimeric vectorRev-Vif-C degrades the protein Rev by the ubiquitination pathway.

FIG. 8 illustrates that the protein encoded by the chimeric vectorRev-Vif-C inhibits the replication of multiple viral strains ofwild-type HIV-1 by inhibiting the nuclear export function of theRev-RRE.

DETAILED DESCRIPTION OF THE PREFERED EMBODIMENT

The present invention will be further described in detail in combinationwith the accompanying drawings and specific embodiments below. Unlessotherwise specified, reagents, equipment and methods used in the presentinvention are conventionally commercial reagents, equipment androutinely used methods in the present technical field.

Embodiment 1 A Construction Model of the Chimeric Vector RBD-Vif-C

As is well-known, the occurring of various tumors such as pancreaticcancer, lung cancer and colorectal cancer is greatly related to mutationof the KRAS gene. A single point mutation of KRAS is sufficient to leadto occurrence of a tumor, wherein the mutation of an amino acid atposition 12 of the KRAS accounts for 98% of the single point mutation ofKRAS. Among the mutation types of the amino acid at position 12, G12Vand G12D mutations are the most significant, which accounts for 30% and51% respectively. Specific degradation of mutant KRAS protein isrealized by a degradation mechanism of target protein induced by Vif,and thus a new type of anti-tumor medicine is developed.

In accordance with the literatures, it is known that there is a KRASbinding domain RBD specifically binding to the mutant KRAS on anN-terminus of a RAF-1 protein. Such binding domain can specifically bindto the mutant KRAS protein in vivo and vitro, and its binding affinityfor GTP-Kras is 100 times different from that for GDP-Kras. Hence,nucleic acid encoding the N-terminus of the Vif protein is replaced withnucleic acid encoding the RBD, and then it is ligated to the vector ofpcDNA3.1 to form a chimeric vector RBD-Vif-C.

Specific steps are provided below:

(1) preparing a chimeric vector, in which nucleic acid encoding an aminoacid sequence at positions 1-79 of the N-terminus of the Vif protein isreplaced with nucleic acid encoding the RAS binding domain RBD which isprovided on a protein structure of Raf and specifically binds toGTP-KRAS, thus a new chimeric vector RBD-Vif-C is constructed;(2) cloning the chimeric vector into the expression vector pcDNA3.1 andperforming a transient expression by transfection;(3) synthesizing nucleic acid encoding a transmembrane peptide segmentPTD and then ligating it to an Escherichia coli expression vector ofpet-32a;(4) performing a PCR amplification using the RBD-Vif-C as a template,ligating the obtained PCR product RBD-Vif-C to the expression vectorpet-32a, and nucleic acid encoding the PTD being located on theN-terminus of the RBD-Vif-C to form a fusion expression vectorPTD-RBD-Vif-C;(5) transforming plasmids of the PTD-RBD-Vif-C into the Escherichia coliBL21(DE3);(6) culturing the transformed Escherichia coli;(7) a single PTD-RBD-Vif-C protein is obtained after a purificationusing a nickel column.

The construction of the vector mentioned in step (1) comprises steps asfollows: synthesizing two pairs of primers according to nucleic acidencoding Vif protein of HIV-1 and KRAS protein respectively, thenperforming the PCR amplification with the aforementioned primers using asequence of PNL4-3 plasmids of HIV-1 virus as the template; then cloningthe PCR amplification product into the pcDNA3.1 by different enzymecutting sites, wherein nucleic acid encoding segment of RBD is insertedinto nucleic acid encoding the N-terminus of the Vif.

Culturing mentioned in step (6) is to inoculate a single colony of thetransformed Escherichia coli to a LB liquid medium containing ampicillinfor cultuting overnight; and then the colony is inoculated to the 37° C.preheated LB liquid medium containing ampicillin with a volume ratio of1:50 for culturing until OD600 reaches 0.6; IPTG is added until having afinal concentration of 0.4 mmol/L, the PTD-RBD-Vif-C protein expresses,and the bacteria is collected after induction.

A method of purification mentioned in step (7) is to wash total bacteriawith PBS Buffer. After being resuspended and ultrasonic treated, alysate supernatant is obtained by a high speed centrifugation, and aneluted protein is collected after an affinity chromatographypurification using the nickel column.

Embodiment 2 the Protein Encoded by the Chimeric Vector RBD-Vif-C canDegrade the KRAS Protein and Inhibit its Signaling Pathway ofPhosphorylation in the Downstream

The mutant KRAS genes, KRAS-G12D and KRAS-G12V, are fusion expressedwith the RFP gene respectively and are cloned into the vector ofpcDNA3.1. The expression of the KRAS can be reflected by observing theexpression of the RFP. Meanwhile, the expression of the KRAS is furtherverified by Western Blot.

(1) Co-transfecting the vector including nucleic acid encoding theKRAS-G12D-RFP with the vector RBD-Vif-C or the KRAS-G12V-RFP with thevector RBD-Vif-C in 293t cells in six-well plates, with a weight ofplasmids of 1 μg; 48 hours after co-transfection, observing theexpression of the RFP; meanwhile collecting a cell lysate for detectingthe expression of the KRAS by the Western Blot.

The experiment shows that the RBD-Vif-C can degrade the mutant KRASprotein.

(2) Co-transfecting the vector including nucleic acid encoding theKRAS-G12D and the plasmid of the RBD-Vif-C in 293t cells in six-wellplates; 48 hours later, collecting the cell lysate for detecting theexpressions of ERK and phosphorylated ERK by the Western Blot.

The experiment shows that the RBD-Vif-C can inhibit its signalingpathway of phosphorylation in the downstream of the KRAS protein bydegrading it.

Embodiment 3 Protein PTD-RBD-Vif-C has a Good Killing Effect on theTumor Cells In Vitro

In consideration of factors such as low efficiency of the transfectionin cell plasmid system of pancreatic cancer, lung cancer and colorectalcancer, and subsequent druggability factor of protein, hence,Escherichia coli is chosen to express the corresponding RBD-Vif-Cprotein while GFP-Vif-C is chosen as a negative control protein, inorder to attempt to verify the inhibition of the expression of KRAS geneby the RBD-Vif-C by means of the action mode of protein directly.

Firstly with reference to the methods from the related literatures, aneffective transmembrane oligopeptide PTD is synthesized and the sequencethereof is shown in the drawings. Then nucleic acid encoding thetransmembrane peptide and the vector RBD-Vif-C are cloned into aprokaryotic expression vector of pET-32a together, expressing theRBD-Vif-C protein by a prokaryotic system. The PTD-RBD-Vif-C proteinwith His-tag is purified by a nickel column and the protein withrelatively high purity can be obtained after several optimizations, asshown in FIGS. 3-13. Further, endotoxin in the protein is removed by themethod of Triton X-114. Since the protein itself has the transmembranepeptide, the purified protein can be directly added into the cellculture supernatant, which provides the subsequent experiments withgreater convenience.

(1) Planking multiple cells into 24-well plates, after the cells beingadhered; adding 4 μg/well of the PTD-RBD-Vif-C protein or thePTD-GFP-Vif-C protein respectively; after treating the cells for 48hours, observing and recording growth states of the various cells with amicroscope.

The experiment shows that PTD-RBD-Vif-C protein can inhibit theproliferation of various tumor cells related to the KRAS.

(2) Adding 0 μg/well, 2 μg/well, 4 μg/well and 8 μg/well of thePTD-RBD-Vif-C protein to the Panc-1 cells and the A549 cells in the24-well plates respectively; collecting the cells after treating themfor 48 hours; labeling flow antibody of Annexin-V FITC and detecting theapoptosis of these two kinds of cells.

The experiment shows the PTD-RBD-Vif-C protein can induce the apoptosisof tumor cells related to the KRAS and has a certain concentrationgradient dependency.

(3) Adding 2 μg/well of the PTD-GFP-Vif-C protein and 2 μg/well of thePTD-RBD-Vif-C protein to the Panc-1 cells and the A549 cells in six-wellplates respectively; after treating them for 12 hours, collecting celllysate for detecting the expression of endogenous KRAS of these twokinds of cells by Western Blot.

The experiment shows that the PTD-RBD-Vif-C protein can inhibit theexpression of the endogenous KRAS gene.

Embodiment 4 PTD-RBD-Vif-C Protein has a Good Anti-Tumor Effect in Mice

Six male Balb/c nude mice of 4-6 months old were ordered from the AnimalExperimental Center of Sun Yat-sen University. They were divided intotwo groups randomly and each group contained three mice. The mice weresubcutaneously inoculated with 1×10⁶ of Panc-1 cells and A549 cellsrespectively to generate tumors. Two weeks later, subcutaneousgeneration of tumor in mice was observed, and the PTD-RBD-Vif-C proteinand the PTD-GFP-Vif-C protein were started to be injected into the mice2-3 times a week. Four weeks later, the mice were executed and tumortissues were taken for observation and were recorded the weight.

The experiment shows that the PTD-RBD-Vif-C protein has a goodanti-tumor effect in mice, especially in the tumor cells of pancreaticcancer and lung cancer.

Embodiment 5 the Acute Toxicity Test of the PTD-RBD-Vif-C Protein inMice

(1) 18 male Balb/c mice of 4-6 months old were ordered from the AnimalExperimental Center of Sun Yat-sen University. They were divided into 3groups randomly and each group contained 6 mice.

(2) The PTD-RBD-Vif-C protein was injected into the mice by means oftail vein injection with an injection dose of 0 mg/kg (PBS), 10 mg/kgand 20 mg/kg respectively, each group contained 6 mice.

(3) Two weeks later, blood samples of the mice were taken for detectionof liver function and renal function, and the experiment result isnoimal.

(4) After execution, each tissue samples of heart, liver, spleen, lungand kidney were taken from the mice to perform HE staining, in order toobserve whether there was a lesion in the organ or not.

The experiment shows that 20 mg/kg dose of protein is non-toxic and safefor the mouse, and such protein has a better druggability.

Embodiment 6 the Construction Model of the Chimeric Vector Rev-Vif-C

Nucleic acid encoding two oligomerization binding domains (amino acidsat positions 9-26 and amino acids at positions 51-65) on the HIV-1 Revgene were respectively cloned to the N-terminus of the Vif gene,replaced nucleic acid encoding the binding sites (amino acids atpositions 1-79) of APOBEC3G, and then were ligated to the vector ofpcDNA3.1 to form three different chimeric vectors, which are named asROL1-Vif-C, ROL2-Vif-C and ROL12-Vif-C respectively. In particular, ROL1represents nucleic acid encoding the oligomerization binding domain ofthe N-terminus of the Rev, i.e. the amino acids at positions 9-26; ROL2represents nucleic acid encoding the oligomerization binding domain ofthe C-terminus of the Rev, i.e. the amino acids at positions 51-65; andROL12 represents nucleic acid encoding the two oligomerization bindingdomains tandem the N-terminus and C-terminus of the Rev, i.e. the aminoacids at positions 9-26 and amino acids at positions 51-65.

Specific steps are as follows:

(1) Respectively replacing nucleic acid encoding the amino acid sequenceat positions 1-79 of the N-terminus of the Vif protein with nucleic acidencoding two oligomerization domains provided on the protein structureof the Rev, thus three new chimeric vectors ROL1-Vif-C (containingnucleic acid encoding the oligomerization domain of the N-terminus ofthe Rev, i.e. the amino acids at positions 9-26), ROL2-Vif-C (containingnucleic acid encoding the oligomerization domain of the C-terminus ofthe Rev, i.e. the amino acids at positions 51-65) and ROL12-Vif-C(containing nucleic acid encoding two oligomerization domains of theN-terminus and C-terminus of the Rev, i.e. the amino acids at positions9-65) were constructed.

(2) Cloning these three chimeric vectors into the expression vectorpcDNA3.1 and performing a transient expression by transfection.

In particular, the construction of the vectors mentioned in step (1)comprises steps as follows: synthesizing 4 pairs of primers according tonucleic acid encoding the Vif protein and the Rev protein of HIV-1respectively, then performing PCR amplification with the aforementionedprimers using the sequence of the PNL4-3 plasmids of HIV-1 virus astemplate; then cloning the PCR amplification product into the pcDNA3.1by different enzyme cutting sites, wherein nucleic acid encoding segmentof the Rev is inserted into nucleic acid encoding the N-terminus of theVif segment.

Embodiment 7 the Protein Encoded by the Chimeric Vector Rev-Vif-CDegrades Rev Protein by the Ubiquitination Pathway

HIV-1 Rev gene was performed a fusion expression with the RFP gene andthey were cloned into the vector of pcDNA3.1, thus the expression of theRev can be reflected by observing the expression of the RFP; meanwhilethe Rev gene was performed the fusion expression with a HA label inorder to further verify the expression of Rev by the Western Blot.

(1) Co-transfecting the vector including nucleic acid encoding theRev-RFP and the vector ROL1-Vif-C (or the ROL2-Vif-C or the ROL12-Vif-C)in the 293t cells in the 24-well plates with an amount of plasmids of1:0, 1:1, 1:2 and 1:3 respectively; 48 hours after transfection,observing the expression of the RFP; meanwhile, co-transfecting thevector including nucleic acid encoding the Rev-HA and the vectorROL1-Vif-C in the 293t cells on the six-well plates with the amount ofplasmids of 1:0, 1:1, 1:2 and 1:3 respectively; 48 hours aftertransfection, collecting the cell lysate for detecting the expression ofRev by the Western Blot.

The experiment shows that the proteins encoded by the three chimericvectors can inhibit the expression of Rev protein and have a certainconcentration gradient dependency.

(2) Co-transfecting 200 ng of the vector including nucleic acid encodingRev-RFP and 200 ng of ROL12-Vif-C vector in the 293t cells in the24-well plates; 24 hours later, adding 10 μM of MG132 to treat thecells; after treating them for 12 hours, observing the expression of theRFP.

(3) Co-transfecting 200 ng of the vector including nucleic acid encodingRev-RFP and 200 ng of ROL12-Vif-C vector in the 293t cells in the24-well plates; 6 hours later, transfecting 50 nM of si-NC, si-ElonginB,si-ElonginC and si-Culin5; 48 hours after transfection, observing theexpression of the RFP.

(4) Co-transfecting 3 μg of Ub-Flag of the plasmid, 3 μg of the vectorincluding nucleic acid encoding Rev-HA and 2 μg of ROL1-Vif-C,ROL2-Vif-C, ROL12-Vif-C vectors respectively in the 293t cells in the 6cm plate; 48 hours after transfection, collecting the cell lysate forCo-IP to enrich the Rev-HA protein, and further detecting theubiquitination of the Rev protein.

The above three experiments show that the protein encoded by thechimeric vectors degrade the Rev protein by the ubiquitination pathwaymediated by the Vif.

Embodiment 8 the Protein Encoded by the Chimeric Vector Rev-Vif-CInhibits the Replication of Various Viral Strains of Wild-Type HIV-1 byInhibiting the Nuclear Export Function of the Rev-RRE

There is a splicing donor SD and a splicing acceptor SA on PDM628. Whenthe Rev protein is not present, the luciferase gene carried by thePDM628 is cleaved, causing a trace expression of luciferase. When twokinds of plasmids co-transfect, the Rev protein is combined with the RREelement and the luciferase gene segment is brought out of the cellnucleus in order to avoid being cleaved by SD and SA, and thus theluciferase expresses massively. Therefore, when the expression of Revprotein is inhibited, the system of nuclear export related to theRev-RRE will be inhibited and the amount of expression of luciferasewill decrease by this time. Based on such principle, it can be judgedwhether the function of the Rev protein is affected or not.

(1) Co-transfecting 10 ng of_(P)DM628 plasmids and 10 ng of Rev plasmidsrespectively in the 293t cells on 96-well plates, then co-transfectingdifferent concentrations of ROL1-Vif-C, ROL2-Vif-C, ROL12-Vif-C and M10plasmids; 48 hours after transfection, collecting the cell lysate todetect the expression of luciferase.

The experiment shows that proteins encoded by three chimeric vectorseach can inhibit the system of nuclear export related to the Rev-RRE,and have a certain concentration gradient dependency. Meanwhile, theeffect of the proteins encoded by the provided chimeric vectors isapparently better than the existing RevM10 mutant.

(2) Co-transfecting 50 ng of PNL4-3 plasmids and differentconcentrations of ROL1-Vif-C, ROL2-Vif-C, ROL12-Vif-C plasmids (50 ng,100 ng, 150 ng) respectively in the 293t cells in 24-well plates; 48hours after transfection, collecting the supernatant to detect theexpression of P24 by ELISA. (B), Co-transfecting 50 ng of PYU-2 plasmidsand different concentrations of ROL1-Vif-C, ROL2-Vif-C, ROL12-Vif-Cplasmids (50 ng, 100 ng, 150 ng) respectively in the 293t cells on the24-well plate; 48 hours after transfection, collecting the supernatantto detect the expression of P24 by ELISA.

The experiment shows that proteins encoded by three chimeric vectorseach can inhibit the replication of various different wild-types HIV-1,and have a certain concentration gradient dependency.

What is claimed is:
 1. A fusion protein comprising a Rev or functionalfragment thereof directly fused to a Vif protein lacking its N-terminalamino acids at positions 1-79 of SEQ ID NO:5.
 2. The fusion protein ofclaim 1, wherein the N-terminal amino acids of Vif at positions 1-79 ofSEQ ID NO: 5 are replaced with at least one oligomerization domain ofthe Rev protein.
 3. The fusion protein of claim 2, wherein the at leastone oligomerization domain of the Rev protein is the N-terminaloligomerization domain of the Rev protein.
 4. The fusion protein ofclaim 3, wherein the N-terminal oligomerization domain of the Revprotein comprises amino acids 9-26 of SEQ ID NO:
 6. 5. The fusionprotein of claim 2, wherein the at least one oligomerization domain ofthe Rev protein is the C-terminal oligomerization domain of the Revprotein.
 6. The fusion protein of claim 5, wherein the C-terminaloligomerization domain of the Rev protein comprises amino acids 51-65 ofSEQ ID NO:
 7. 7. The fusion protein of claim 2, wherein the at least oneoligomerization domain of the Rev protein is both the N-terminal and theC-terminal oligomerization domains of the Rev protein.
 8. The fusionprotein of claim 7, wherein the N-terminal and C-terminaloligomerization domains of the Rev protein comprise amino acids 9-65 ofSEQ ID NO:
 8. 9. A composition comprising the fusion protein of claim 1and a pharmaceutically acceptable carrier.
 10. A nucleic acid moleculeencoding the fusion protein of claim
 1. 11. An expression vectorcomprising the nucleic acid molecule of claim
 10. 12. A host cellcomprising the expression vector of claim
 11. 13. A method for making afusion protein comprising a Rev or functional fragment thereof directlyfused to a Vif protein lacking its N-terminal amino acids at positions1-79 of SEQ ID NO:5, said method comprising: culturing the host cell ofclaim 12 under conditions that result in the production of a fusionprotein comprising a Rev or functional fragment thereof directly fusedto a Vif protein or a functional fragment thereof; and isolating thefusion protein from the host cell culture.
 14. A fusion proteincomprising a Raf or functional fragment thereof directly fused to a Vifprotein lacking its N-terminal amino acids at positions 1-79 of SEQ IDNO:5.
 15. The fusion protein of claim 14, wherein the N-terminal aminoacids of Vif at positions 1-79 of SEQ ID NO: 5 are replaced with abinding domain of an N-terminus of the Raf protein which canspecifically bind to GTP-Kras.
 16. A composition comprising the fusionprotein of claim 14 and a pharmaceutically acceptable carrier.
 17. Anucleic acid molecule encoding the fusion protein of claim
 14. 18. Anexpression vector comprising the nucleic acid molecule of claim
 17. 19.A host cell comprising the expression vector of claim
 18. 20. A methodfor making a fusion protein comprising a Raf or functional fragmentthereof directly fused to a Vif protein lacking its N-terminal aminoacids at positions 1-79 of SEQ ID NO:5, said method comprising:culturing the host cell of claim 19 under conditions that result in theproduction of a fusion protein comprising a Raf or functional fragmentthereof directly fused to a Vif protein or a functional fragmentthereof; and isolating the fusion protein from the host cell culture.